Early diagnosis of infection is crucial to patient survival and public health. This proposal will address this imperative by developing an innovative real-time PCR lab-on-a-chip technology to detect microbial DNA in clinical samples. This novel PCR platform will be tested on two urgent challenges in infectious diseases, the need to establish an early diagnosis of candidemia in high risk patients and the need for a definitive diagnostic test to detect community-acquired mycoplasmal pneumonia. This proposal reflects a synergistic collaboration involving basic and clinical investigators at Duke University and scientists at Advanced Liquid Logic, Inc., who are pioneering the development of a nanodroplet thermocycler for real-time PCR. We will apply this digital microfluidics technology to develop a PCR method to detect DNA of Candida species or Mycoplasma pneumoniae in blood or respiratory specimens, respectively. Addressing the focus on nosocomial bloodstream infections and community acquired pneumonia, we propose to investigate (1) opportunistic candidemia in neonatal, pediatric, and adult patients, including transplant patients, and (2) acute mycoplasmal pneumonia in children and adults. We will implement retrospective and prospective clinical studies to evaluate critically the diagnostic capability of this method. The project involves three interrelated specific aims (1) to develop an integrated nanodroplet thermocycler for real-time PCR, (2) to evaluate the ability of this on-chip PCR to diagnose candidemia in high risk patients, and (3) to evaluate the ability of this on-chip PCR to diagnose community-acquired mycoplasmal pneumonia. The proposed method is extremely rapid and amenable to implementation at the bedside, stat laboratory, or physician's office. This project may ultimately lead to an integrated and portable lab-on-a-chip diagnostic platform for the fully automated, rapid detection of a panel of infectious diseases by real-time PCR.